The present invention relates generally to the amplification of nucleic acids. More specifically, the present invention facilitates the amplification of mRNA for a variety of end uses.
Many biological functions are accomplished by altering the expression of various genes through transcriptional (e.g. through control of initiation, provision of RNA precursors, RNA processing, etc.) and/or translational control. For example, fundamental biological processes such as cell cycle progression, cell differentiation and cell death, are often characterized by the variations in the expression levels of a group of genes.
Gene expression is also associated with pathogenesis. For example, the lack of sufficient expression of functional tumor suppressor genes and/or the over expression of oncogene/protooncogenes could lead to tumorgenesis (Marshall, Cell, 64: 313-326 (1991); Weinberg, Science, 254: 1138-1146 (1991), incorporated herein by reference for all purposes). Thus, changes in the expression levels of particular genes (e.g. oncogenes or tumor suppressors) serve as signposts for the presence and progression of various diseases.
Highly parallel methods of monitoring the expression of a large number of genes in a biological sample are a valuable research and diagnostics tool. However, the amount of starting material that can be obtained from a given source is often limited and it is useful to amplify genetic material prior to analysis. Methods of amplification that allow analysis of a sample that may be too small for analysis without amplification facilitate the analysis of gene expression in small samples and possibly in a single cell.
The present invention provides methods for monitoring expression of a plurality of genes in a cell, one or more cells, a small population of cells or a small amount of biological sample. Preferred methods entail amplifying a population of nucleic acids derived from a population of fewer than 1000 cells.
The invention provides methods for the amplification of nucleic acids that may comprise synthesizing double-stranded DNA from a single-stranded DNA population, and producing multiple copies of RNA from the double-stranded DNA. The method further comprises a second round of amplification comprising: synthesizing single stranded DNA from the multiple copies of RNA and producing multiple copies of RNA from the DNA. The second round of amplification may be repeated.
More specifically, in one preferred embodiment (see, FIG. 1), the method comprises contacting a mRNA having a poly dA tail with a primer comprising poly d(T) and a second sequence; generating a first cDNA strand from the mRNA strand by extending the primer by reverse transcriptase and the appropriate nucleotides under the appropriate conditions, which creates a RNA:DNA duplex; digesting the RNA with RNaseH; forming a double stranded DNA; denaturing the double stranded DNA to form a single stranded DNA and adding a promoter to the single stranded DNA, the promoter comprising a complement to the second sequence and a RNA polymerase promoter sufficient to form a functional promoter when the promoter is hybridized to the single stranded DNA; forming a double stranded DNA promoter region by adding the appropriate reagents; and, producing multiple copies of RNA from the DNA strand comprising the promoter, the RNA copies comprising a complement to at least a portion of the second sequence. Preferably, the promoter is blocked from 3xe2x80x2 extension.
The method further comprises a second round of amplification (see, FIG. 1, steps 7-10), comprising contacting the multiple RNA copies with random primers; generating a first strand cDNA from the RNA by extending the primers with reverse transcriptase and the appropriate nucleotides under the appropriate conditions, which creates a RNA:DNA duplex; digesting the RNA:DNA duplex with for example, RNaseH, to form a single stranded DNA and adding a primer to the single stranded DNA, the primer comprising a complement to the second sequence and an RNA polymerase promoter sufficient to form a functional promoter when the primer is hybridized to the single stranded DNA, forming a double stranded DNA promoter region by adding the appropriate reagents; and, producing multiple copies of RNA from the DNA strand comprising the promoter. Preferably, the promoter is blocked from 3xe2x80x2 extension.
In another embodiment of the invention (see FIG. 2), the method comprises contacting a mRNA having a poly dA tail with a primer comprising poly d(T) and a second sequence that is a promoter; generating a first cDNA strand from the mRNA strand by extending the primer by reverse transcriptase and the appropriate nucleotides under the appropriate conditions, which creates a RNA:DNA duplex; denaturing the RNA:DNA duplex; contacting the resulting single stranded DNA with random primers and extending the primers with DNA polymerase and the appropriate nucleotides under the appropriate conditions; which creates a double stranded DNA with a promoter region; and, producing multiple copies of RNA from the DNA strand comprising the promoter.
The method further comprises a second round of amplification, (see, FIG. 2 steps 5-10 comprising contacting the multiple RNA copies with random primers; generating a first cDNA strand from the RNA by extending the primers with reverse transcriptase and the appropriate nucleotides under the appropriate conditions, which creates a RNA:DNA duplex; denaturing the RNA:DNA duplex to form a single stranded DNA and contacting the single stranded DNA with a primer comprising poly d(T) and a second sequence that is a promoter, forming a double stranded DNA by extending the primer with DNA polymerase and the appropriate nucleotides under the appropriate conditions; forming a double stranded DNA promoter region by adding the appropriate reagents; and, producing multiple copies of RNA from the DNA strand comprising the promoter.
In another embodiment the second round of amplification is repeated at least one time.
Among other factors, the present invention provides new methods for amplification of nucleic acids. The methods are particularly useful for amplification of small samples such as a sample derived from 1000 or fewer cells. Additionally, in one embodiment, the present invention is an amplification method in which a promoter is protected from degradation throughout the method.
The present invention also preferably provides methods, which may further comprise contacting the multiple copies of RNA with a solid support comprising nucleic acid probes, and detecting the presence or absence of hybridization of the RNA to the nucleic acid probes on the solid support. In a preferred embodiment, the solid support, which may comprise nucleic acid probes, can be selected from the group consisting of a nucleic acid probe array, a membrane blot, a microwell, a bead, and a sample tube.
In yet another preferred embodiment, the invention relates to a kit comprising reagents and instructions for the amplification of mRNA. Preferably, the kit includes a reaction vessel containing one or more reagents in concentrated form, where the reagent may be an enzyme or enzyme mixture. The kit also includes a container, instructions for use, a primer which comprises a poly d(T) sequence operably linked to a second sequence, and a primer comprising the second sequence or its equivalent operably linked to a promoter sequence. Preferably, the primer comprising the promoter is blocked from extending in the 3xe2x80x2 direction.
In yet another preferred embodiment, the invention relates to a kit comprising reagents and instructions for the amplification of mRNA. Preferably, the kit includes a reaction vessel containing one or more reagents in concentrated form, where the reagent may be an enzyme or enzyme mixture. The kit also includes a container, instructions for use, and a primer which comprises a poly d(T) sequence operably linked to a second sequence comprising a promoter.